JP2010519920A - イオン性洗剤によるポリメラーゼ安定化 - Google Patents
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Abstract
両性イオン性洗剤を含む組成物に関する。本発明は、また、(i)核酸ポリメラーゼ活性を有する酵素、(ii)不活性タンパク質および、(ii)両性イオン性洗剤を含む組成物に関する。本発明は更に、(a)ポリメラーゼ活性、核酸鋳型、両性イオン性洗剤、緩衝液、塩、ヌクレオチドおよび不活性タンパク質を反応混合物に提供する工程および、(b)反応混合物を核酸合成可能な温度でインキュベートする工程を含む酵素的核酸合成方法に関する。
【選択図】なし
Description
本発明は、酵素、特にポリメラーゼを安定化する組成物および方法に関する。本発明者らは、驚くべきことに、(a)核酸ポリメラーゼ活性を有する酵素、(b)不活性タンパク質および、(c)イオン性洗剤を含む組成物が、酵素、特にポリメラーゼにとって理想的な安定剤であることを見出した。
不活性タンパク質とは、酵素活性または当該の酵素反応を妨げない、天然に生じる、または合成のペプチドもしくはポリペプチド、またはこれらの混合物を意味する。本発明の範囲を制限しない例は、グロブリン、アルブミン、コラーゲンおよびそれらの誘導体である。
核酸ポリメラーゼ活性を有する酵素とは、リボヌクレオシドトリホスフェートまたはデオシキヌクレオシドトリホスフェートから核酸鎖(例えば、RNAまたはDNA)を合成する酵素の能力を意味する。DNAポリメラーゼは、DNAを合成し、一方、RNAポリメラーゼは、RNAを合成する。
実施例1:実施例1は、図1中の好ましい両性イオン性洗剤を示す。
Claims (18)
- a.核酸ポリメラーゼ活性を有する酵素、
b.不活性タンパク質、および
c.両性イオン性洗剤、
を含む組成物であって、核酸ポリメラーゼが組み換え型である組成物。 - 酵素はTaq DNAポリメラーゼではない、請求項1に記載の組成物。
- 非イオン性洗剤を含まない、請求項1または2に記載の組成物。
- 不活性タンパク質は、天然または合成のペプチド、ポリペプチド、グロブリン、コラーゲンならびにそれらの誘導体、および血清アルブミンならびにその誘導体およびフラグメントの群から選択される、請求項1〜3に記載の組成物。
- 不活性タンパク質は、ウシ血清アルブミン(BSA)であり、かつ前記タンパク質は0.01mg/ml超、0.05mg/ml超、および0.1mg/ml超の群から選択される濃度で存在する、請求項4に記載の組成物。
- 両性イオン性洗剤は、0.0005容量%〜5.0容量%の範囲の濃度で存在する、請求項1〜5に記載の組成物。
- 両性イオン性洗剤は、0.001容量%〜0.4容量%の範囲の濃度で存在する、請求項6に記載の組成物。
- 両性イオン性洗剤は、3−〔(3−コールアミドプロピル)ジメチルアンモニオ〕−1−プロパンスルホネート(CHAPS)、3−〔(3−コールアミドプロピル)ジメチルアンモニオ〕−2−ヒドロキシ−1−プロパンスルホネート(CHAPSO)、N−(アルキルC10〜C16)−N,N−ジメチルグリシンベタイン(EMPIGEN BB)、カプリリルスルホベタイン(SB3-10)、3−〔N,N−ジメチル(3−ミリストイルアミノプロピル)アンモニオ〕プロパンスルホネート(アミドスルホベタイン−14;ASB-14)、N−テトラデシル−N,N−ジメチル−3−アンモニオ−1−プロパンスルホネート(3-14 Detergent;ZWITTERGENT)、N−ドデシル−N,N′−ジメチル−3−アンモニオ−1−プロパンスルホネート、N−オクタデシル−N,N−ジメチル−3−アンモニオ−1−プロパンスルホネート、N−デシル−N,N−ジメチル−3−アンモニウム−1−プロパンスルホネート、Mirataine CB、Mirataine BB、Mirataine CBR、Mirataine ACS、Miracare 2MHTおよび、Miracare 2MCAの群から選択される、請求項1〜7に記載の組成物。
- 両性イオン性洗剤は、3−〔(3−コールアミドプロピル)ジメチルアンモニオ〕−1−プロパンスルホネート(CHAPS)、3−〔(3−コールアミドプロピル)ジメチルアンモニオ〕−2−ヒドロキシ−1−プロパンスルホネート(CHAPSO)および、N−(アルキルC10〜C16)−N,N−ジメチルグリシンベタイン(EMPIGEN BB)の群から選択される、請求項8に記載の組成物。
- 組成物は反応緩衝液であり、かつ前記組成物は、緩衝剤、一価塩、二価カチオンおよびヌクレオチドの群から選択される物質を追加的に含む、請求項1〜9のいずれかに記載の組成物。
- 組成物は保存緩衝液であり、かつ前記組成物は、緩衝剤、還元剤、キレート剤、還元剤およびグリセロールの群から選択される物質を追加的に含む、請求項1〜9のいずれかに記載の組成物。
- 緩衝剤は、酢酸塩緩衝液、硫酸塩緩衝液、リン酸塩緩衝液、MOPS、HEPESおよびトリス−(ヒドロキシメチル)アミノメタン(TRIS)の群から選択される、請求項10または11に記載の組成物。
- 保存緩衝液中または反応緩衝液中の核酸ポリメラーゼの安定化のための
a.不活性タンパク質、および
b.両性イオン性洗剤、
を含む組成物の使用。 - PCR反応混合物または核酸ポリメラーゼ保存緩衝液へ
a.不活性タンパク質、および
b.両性イオン性洗剤、
を含む組成物を添加することを含む、核酸ポリメラーゼの安定化方法。 - a.核酸ポリメラーゼ活性、核酸鋳型、両性イオン性洗剤、緩衝液、塩、ヌクレオチドおよび不活性タンパク質を反応混合物に提供する工程;
b.反応混合物を核酸合成可能な温度でインキュベートする工程
を含む酵素的核酸合成方法。 - 酵素的核酸合成は、DNAシークエンシング、プライマー伸長アッセイ、DNA増幅およびRNAのDNAへの逆転写の群から選択される方法において実施される、請求項15に記載の方法。
- a.核酸ポリメラーゼ活性を有する酵素、
b.不活性タンパク質、および
c.両性イオン性洗剤、
を含むキットであって、核酸ポリメラーゼが組み換え型であるキット。 - 請求項15または16の方法を実施するための請求項17に記載のキット。
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US90503107P | 2007-03-06 | 2007-03-06 | |
EP07103648A EP1970440A1 (en) | 2007-03-06 | 2007-03-06 | Polymerase stabilization by ionic detergents |
PCT/EP2008/052718 WO2008107473A1 (en) | 2007-03-06 | 2008-03-06 | Polymerase stabilization by ionic detergents |
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JP2010519920A true JP2010519920A (ja) | 2010-06-10 |
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JP2009552209A Pending JP2010519920A (ja) | 2007-03-06 | 2008-03-06 | イオン性洗剤によるポリメラーゼ安定化 |
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US (1) | US20100099150A1 (ja) |
EP (2) | EP1970440A1 (ja) |
JP (1) | JP2010519920A (ja) |
WO (1) | WO2008107473A1 (ja) |
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- 2008-03-06 US US12/529,434 patent/US20100099150A1/en not_active Abandoned
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EP1970440A1 (en) | 2008-09-17 |
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